Methods of identifying kinetically stable proteins

ABSTRACT

The present invention provides a fast and efficient means for identifying kinetically stable proteins. As used herein the term “kinetically stable protein” means a protein that is trapped in a specific conformation due to an unusually high unfolding barrier that results in very slow unfolding rates. The present inventors are the first to discover the existence of a correlation between kinetic stability and SDS-induced denaturation. Thus, the invention provides methods for identifying kinetically stable proteins comprising the step of testing the proteins for resistance to denaturation by SDS. In one embodiment, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is one simple method for quickly identifying and selecting kinetically stable proteins. In another embodiment a two-dimensional SDS-PAGE provides a high throughput method for quickly identifying kinetically stable proteins in a sample.

RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.10/924,235, filed on Aug. 23, 2004, which claims the benefit of U.S.Provisional Application No. 60/496,778, filed on Aug. 21, 2003. Theentire teachings of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant, NSFMCB-9984913, from the National Science Foundation. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Kinetic stability is a poorly understood property of a select group ofnaturally occurring proteins that are trapped in their nativeconformations by an energy barrier, and consequently are resistant tounfolding. Kinetic stability can be best explained by illustrating theunfolding process as a simple equilibrium reaction between two proteinconformations, the native folded state (N) and the unfolded state (U),separated by a higher energy transition state (TS) (FIG. 1). Since theheight of the TS barrier determines the rate of folding and unfolding,kinetically stable proteins possess an unusually high energy TS, whichresults in extremely slow unfolding rates that virtually trap theprotein in its native state (FIG. 1A). Even though the overall change inGibbs free energy (ΔG) may be favorable for unfolding under extremesolvent conditions, such as high concentrations of denaturant, the highactivation energy of the TS significantly slows down the unfolding rate(FIG. 1B). It has been suggested that the presence of a high kineticenergy barrier separating the folded and unfolded states is anevolutionary feature intended to allow proteins to maintain activity inthe extreme conditions they may encounter in vivo (1). The examples ofthe kinetically stable proteins α-lytic protease (extracellular enzyme)(1), Escherichia coli OmpA (bacterial membrane protein) (2), andpyrrolidone carboxyl peptidase (hyperthermophilic protein) (3),illustrate the kinetic adaptation of proteins that must retain enzymaticfunction in conditions where degradation might easily take place. Inaddition, thermodynamic stability alone does not fully protect proteinsthat are susceptible to irreversible denaturation and aggregationarising from partially denatured states that become transientlypopulated under physiological conditions (4). Therefore, the developmentof a high kinetic energy barrier to unfolding may serve to protectsusceptible proteins against such harmful conformational “side-effects”.

The physical basis for kinetic stability is poorly understood and nostructural consensus has been found to explain this phenomenon. Inprevious studies, the addition. of hydrophobic residues on the proteinsurface (5), the engineering of disulfide bonds (6), and theintroduction of metal-binding sites (7) have been shown to increasekinetic stability. A connection between kinetic stability and oligomericquaternary structure has also been proposed (8). In the case of somehyperthermophilic proteins, electrostatic interactions have beensuggested to be a major factor in their slow unfolding due to theformation of ion pairs (9, 10). However, there is evidence that somekinetically stable proteins retain their slow unfolding rate even at lowpH, where electrostatic interactions should be significantly weakened(3, 11). Thus, it appears that no common structural feature exists toexplain kinetic stability, and perhaps this property may be achieved bydifferent means, depending on the individual protein.

Under native conditions, kinetically stable proteins have limited accessto partially and globally unfolded conformations (12). These propertiesimpart a strong proteolytic resistance by reducing the occurrence ofaccessible conformations susceptible to proteolytic attack (12, 13).Some kinetically stable proteins have also been found to be resistant todenaturation by sodium dodecyl sulfate (SDS). Among them are the β-sheetproteins streptavidin (14), transthyretin (15), P22 tailspike protein(16), and the e-coli membrane protein, OmpA (2).

The ability to quickly and easily identify kinetically stable proteinswould have a myriad of applications in the biotechnology industry,pharmaceutical industry, and in basic life science research.

SUMMARY OF THE INVENTION

The present invention provides a fast and efficient means foridentifying kinetically stable proteins. As used herein the term“kinetically stable protein” means a protein that is trapped in aspecific conformation due to an unusually high unfolding barrier thatresults in very slow unfolding rates. The present inventors are thefirst to discover the existence of a correlation between kineticstability and SDS-induced denaturation. Thus, the invention providesmethods for identifying kinetically stable proteins comprising the stepof testing the proteins for resistance to denaturation by SDS.

In one embodiment, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) isone simple method for quickly identifying and selecting kineticallystable proteins. In another embodiment, a diagonal two-dimensional gelelectrophoresis (2DE or 2D SDS-PAGE) method is used in which an SDS-PAGEseparation is performed in both dimensions of the analysis.

The methods of the invention have the advantage that proteins can beeasily tested for kinetic stability without having to carry outunfolding experiments. Also, only microgram amounts of sample areneeded, and the method is potentially suitable for identifyingkinetically stable proteins present in cell extracts without need forpurification. From an application perspective, this assay has thepotential of being adaptable for various high-throughput applicationsfor identification of kinetically stable proteins from complex mixtures.The method has many potential applications in the fields of basicbioscience, pharmaceuticals, medical diagnostics and engineering.

The methods of the invention may be useful for various high throughputapplications to enhance the kinetic stability of proteins of interest.This could lead to proteins with greater shelf life and/or decreasedtendency to aggregate, consistent with the suggestion that thedeterioration of an energy barrier between native and pathogenic statesas a result of mutation may be a key factor in the misfolding andaggregation of some proteins linked to amyloid diseases (4, 18). Suchprotein misfolding diseases include but are not limited to Alzheimer'sdisease, Parkinsons, prior-related encephalopathies, amyotrophic lateralsclerosis, and type II diabetes.

Another important application of the methods of the invention involvesevaluating the kinetically stable proteome of healthy human plasma andcomparing it to that associated with various diseases to identifyimportant biomarkers for early diagnosis of these diseases in humans.Combination of this approach with Western blotting protocol for specificprotein quantification would provide a valuable diagnostic tool fordisease detection.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings.

FIG. 1 is a free energy diagram to illustrate the higher unfoldingenergy barrier for a kinetically stable protein under native (A) anddenaturing (B) conditions as compared to that of a normal protein(represented by the dash line).

FIG. 2 is an SDS-PAGE assay of SDS-resistant proteins (A) and the nonSDS-resistant control group (B). Identical protein samples were eitherunheated (U) or boiled (B) for 10 min immediately prior to loading ontothe gel.

FIG. 3 shows fluorescence-detected unfolding kinetics of theSDS-resistant proteins upon incubation in 6.6 M guanidine hydrochloride(GdnHCl) at 20° C. Relative fluorescence was obtained by dividing eachdata point by the first point of the native protein baseline. No missingamplitude was observed, indicating that the observed kinetic traceaccounts for the full unfolding transition.

FIG. 4 shows the unfolding rate of the SDS-resistant (A) and nonSDS-resistant control group (B) under native-like conditions shown byextrapolating the unfolding rate determined at different concentrationof GdnHCl to 0 M. The y-intercept of each extrapolation curve indicatesthe native protein's unfolding rate (Table 2)

FIG. 5 shows the proteolytic resistance of the SDS-resistant proteins(A) and proteolytic susceptibility of the non-resistant control group(B) determined by incubating them for 48 hours with Proteinase K using aprotease:protein ratio of 1:100 (w/w) and boiling the samples prior toloading onto the gel.

FIG. 6 are ribbon diagrams of the SDS-stable proteins revealing the highcontent of oligomeric β-sheet structure. Coordinates were obtained fromthe Protein Data Bank using the following PDB codes: SOD-1SPD; SVD-1SWU,TTR-1GKE, TSP-1TYU, CPAP-1YAL, PAP-1PPN, AVD-1RAV, SAP-1SAC

FIG. 7A–C is a diagonal 2D SDS-PAGE separation of (A) non-SDS resistantproteins only, (B) SDS-resistant proteins only, and (C) a mixture ofSDS-resistant and non-SDS resistant proteins.

FIG. 8 is a diagonal 2D SDS-PAGE separation of the cell lysate from E.coli.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used herein: ADH, Yeast AlcoholDehydrogenase; AVD, Avidin; B2M, Beta2-microglobulin; BLA, BovineAlpha-Lactalbumin; ConA, Concanavalin A; CPAP, Chymopapain; GAPDH,Glyceraldehyde 3-phosphate Dehydrogenase; GdnHCl, guanidinehydrochloride; MW, molecular weight; PAP, Papain; PB, phosphate buffer;SAP, Serum Amyloid P; SDS-PAGE, sodium dodecyl sulfate-polyacrylamidegel electrophoresis; SOD, Copper/Zinc Superoxide Dismutase; SVD,Streptavidin; TSP, P22 Tailspike protein; TIM, Triosephosphate Isomerasefrom porcine muscle; TTR, Transthyretin; Tris,2-amino-2-hydroxymethyl-1,3-propanediol.

The invention provides a method for identifying a kinetically stableprotein by testing the protein for resistance to denaturation by SDS. Inone embodiment involving SDS-PAGE, kinetically stable proteins areidentified by a method comprising the steps of:

-   -   a) providing at least one pair of substantially identical        protein samples;    -   b) adding SDS to each protein sample;    -   c) boiling only one of the protein samples of the pair of        protein samples;    -   d) conducting SDS-polyacrylamide gel electrophoresis on the pair        of protein samples; and    -   e) comparing the migration on the gel of proteins contained in        the boiled protein sample to the migration on the gel of        proteins contained in the unheated protein sample.        Proteins that migrate to the same location on the gel regardless        of whether or not the sample was boiled are classified as not        being stable to SDS and are therefore not kinetically stable.        Proteins that exhibit a slower migration when the sample is not        boiled are at least partially SDS resistant and are considered        to be kinetically stable. As used herein the term “substantially        identical protein samples” means that each of the protein        samples are collected from the same or similar source such as        from the same protein stock solution or protein library, wherein        it is likely that a high percentage of the same proteins are        present in each sample of the protein sample pair.

In yet another embodiment, a fluorescence-based assay may be used totest a protein's resistance to SDS denaturation. This assay may beparticularly useful as the basis for a high throughput screening assay.In one embodiment a fluorescence-based assay for measuring SDSresistance comprises the steps of: a) generating a library of proteinsby a display methodology using a well plate format; b) contacting thelibrary of displayed proteins with SDS; c) washing unbound SDS from theproteins; d) contacting the protein library with a fluorescent probecapable of binding to an SDS-protein complex; and e) determining theintensity of fluorescence in each well. Kinetically stable proteins willbe identified by their lack of fluorescence. This is because only theproteins that bind SDS will be fluorescent and the intensity offluorescence may be used to quantify the extent of kinetic stability.Display methodologies are well known in the art and include phagedisplay, yeast protein display, antibody display and bacterial display.Various methods of phage display are described in U.S. Pat. Nos.5,223,409 and 6,057,098, incorporated herein by reference. Yeast proteindisplay is described in Border and Winthrop, Nature Biotech. 15:553(1997). One skilled in the art recognizes that there are a variousprotocols for determining a protein's resistance to SDS denaturation andthat all such protocols are useful in the methods of the presentinvention.

In another aspect, the invention provides a method of identifying thepresence of kinetically stable proteins in a library of proteinsgenerated by any means, comprising the step of testing proteins in theprotein library for resistance to denaturation by SDS. For example,either an SDS-PAGE assay or fluorescence-based assay, both as describedabove, or the 2D SDS-PAGE assay as described below, may be used toidentify kinetically stable proteins in the protein library. The methodsof the invention are particularly useful in improving processesinvolving the manipulation and engineering of proteins by providing arapid means for identifying kinetically stable proteins in the librariesof known proteins, random proteins, mutagenized proteins, chimericproteins, fusion proteins, and chimeric fusion proteins often producedin the course of such protein manipulation or engineering.

A protein library may be the result of, for example, rational proteindesign, site-directed mutagenesis, directed evolution, protein genesis,and gene shuffling, or the protein library may be a combinatorial arrayof proteins. The fluorescence assay and the 2-dimensional SDS PAGE assayof the present invention are particularly useful for high-throughputscreening to identify kinetically stable proteins present in suchprotein libraries.

In one embodiment, the present invention may be used to identifykinetically stable chimeric polypeptides generated from a library ofchimeric polynucleotides prepared in accordance with known techniquesfor producing libraries of chimeric polynucleotides and combinatorialchimeric polynucleotides (e.g. rational protein design, protein genesis,directed evolution, site-directed mutagenesis and gene shuffling. Asused herein, a “polynucleotide” is a polymeric chain of nucleotides(e.g., a gene, gene fragment, cDNA, mRNA), and a “polypeptide” is apolymeric chain of amino acids (e.g., a protein). “Chimericpolynucleotides” are polynucleotides that contain regions derived fromtwo or more parent genes as opposed to site-directed mutagenized DNAcomprising only point mutations or insertion and deletion mutations(“indels”). Chimeric polynuceotides are useful in techniques such as“gene shuffling” (see, e.g. Crameri, A., et al., Nature 391(6664):228–291 (1998)) and other processes aimed at engineering proteinswith novel properties (see also U.S. Pat. No. 5,223,409).

The invention is particularly suited for application to the methods ofgenerating, by directed evolution, chimeric polypeptides as described incopending U.S. application Ser. No. 10/712,806, filed on Nov. 13, 2003and copending U.S. application Ser. No. 10/138,183, filed on May 2,2003, both of which are incorporated herein by reference in theirentirety. These applications pertain to methods for generating chimericpolynucleotides, such as polynucleotides encoding polypeptides(“chimeric polypeptides”), using directed evolution of a basis set ofpolynucleotides. A “basis set” is a group of 2 or more polynucleotides,preferably greater than or equal to 3 polynucleotides, such as between 3and 12 polynucleotides, inclusive, or more; the basis set ofpolynucleotides is used as the starting materials for the directedevolution. The present invention is particularly useful in identifyingkinetically stable chimeric polypeptides resulting from the directedevolution process described therein.

The invention is also suited for identifying kinetically stable proteinsand polypeptides produced by the various methods described in thefollowing patent applications: U.S Provisional Patent Applications Nos.60/445,689, 60/445,704, 60/445,703 (all filed on Feb. 6, 2003) and60/474,063 (filed on May 29, 2003), all incorporated herein by referencein their entirety. The methods described in these patent applicationsrelate to generating libraries of chimeric oligonucleotides,combinatorial chimeric oligonucleotides and oligonucleotides havingpoint mutations, insertions and deletions using polymerase-basedprocedures, for expression in a host cell. The resulting libraries ofpolypeptides and proteins may then be conveniently screened forkinetically stable proteins using the methods of the present inventions.

Thus the invention provides methods of identifying kinetically stablechimeric polypeptides produced from a library of chimericpolynucleotides generated by directed evolution comprising the steps of:a) generating a library of chimeric polynucleotides by directedevolution; b) expressing the library of chimeric polynucleotides in ahost cell; and c) screening the resulting chimeric polypeptides forresistance to denaturation by SDS. In one embodiment, the screening stepcomprises assaying the chimeric polypeptides for resistance todenaturation by SDS comprising the steps of:

a) providing at least one pair of substantially identical chimericpolypeptide samples derived from a chimeric polypeptide library, eachsample comprising at least one polypeptide in combination with SDS;

b) boiling only one of the chimeric polypeptide samples of the pair ofsamples;

c) conducting SDS-polyacrylamide gel electrophoresis on the pair ofchimeric polypeptide samples; and

d) comparing the migration of polypeptides contained in the boiledchimeric polypeptide sample to the migration of polypeptides containedin the unheated chimeric polypeptide sample. Kinetically stablepolypeptides present in the cell extract of the host cells used toexpress the polypeptides can be identified using an SDS-PAGE assaywithout the need for further purification steps.

In addition to identifying chimeric polypeptides, the invention alsoprovides methods for identifying kinetically stable mutated proteins ina library of mutated proteins generated by site-directed mutagenesis ofpolynucleotides (e.g. by insertions, deletions and point mutations ofpolynucleotides) In one embodiment, the invention provides a method foridentifying kinetically stable mutated proteins in a library of mutatedproteins produced by site-directed mutagenesis comprising the steps of:

-   -   a) generating by site-directed mutagenesis, a library of mutated        polynucleotides;    -   b) expressing the library of mutated polynucleotides in host        cells; and    -   c) screening the resulting mutated polypeptides expressed in the        host cells for resistance to denaturation by SDS. Any of the        methods for testing a protein's resistance to SDS denaturation        as described herein are useful for screening the resulting        mutated polypeptides expressed in the host cells for resistance        to denaturation by SDS.

In yet another aspect of the invention, a diagonal 2DE method in whichan SDS-PAGE separation is performed in both dimensions is used toidentify kinetically stable proteins. By using this methodology theinventors are able to translate their one-dimensional SDS resistanceassay described above to 2D SDS-PAGE for rapid and high throughputdetermination of kinetic stability. In accordance with this method ofthe invention, a diagonal 2DE method is used in which an SDS-PAGEseparation of a protein sample is performed in both dimensions of theanalysis. As described in detail in the Examples, protein mixtures arefirst separated according to molecular weight on a narrow strip of gel.The gel strip is then incubated in SDS-PAGE treatment buffer whilebriefly immersed in a boiling water bath before being transferred onto alarger gel for the second dimension run. One exemplary 2D SDS-PAGE assayof the invention for use in identifying kinetically stable proteinscomprises the steps of: a) electrophoretically separating the componentsof a sample according to weight on a strip of an SDS-polyacrylamide gelin a first dimension; b) incubating the gel from step (a) in SDS-PAGEtreatment buffer while immersing in boiling water; (c) transferring thegel from step (b) to a second, larger, SDS-polyacrylamide gel forelectrophoretic separation in the second dimension; and d) identifyingthe kinetically stable proteins on the second dimension gel. Kineticallystable proteins are identified as those proteins falling off of thediagonal in the second dimension gel after electrophoretic separation inthe second dimension has occurred.

The two dimensional assay of the invention for identifying kineticallystable proteins may be used in place of, or in addition to, the onedimensional assays described herein. For example, the two dimensionalassay of the invention may be used in methods of identifying thepresence of kinetically stable proteins in a library of proteinsgenerated by any means; methods of high throughput screening foridentifying kinetically stable chimeric proteins including thoseresulting from directed evolution; and methods of identifying stablemutated proteins in a library of mutated proteins generated by any meansincluding site-directed mutagenesis of polynucelotides; all as describedabove.

One skilled in the art will appreciate the many advantages that themethods of the invention provides in addition to those described above.For example, the methods of the invention are useful in protein andenzyme engineering technologies (e.g. rational protein design, proteingenesis, directed evolution, site-directed mutagenesis and geneshuffling) for the production of industrial proteins and enzymes such asdetergent enzymes, enzymes useful for neutralizing contaminants andenzymes useful as fuel additives. Likewise, the methods of the inventionare useful in protein engineering technologies for the production ofproteins and enzymes useful in the food and life sciences industriessuch as primary and secondary metabolites useful in the production ofantibiotics, proteins and enzymes for the food industry (bread, beer),and combinatorial arrays of proteins for use in generating multipleepitopes for vaccine production. The methods of the invention are alsoparticularly useful in the design and development of diagnostics ortherapeutics where protein stability is a desired or necessarycharacteristic of the diagnostic reagent or the embodiments in which theability to quickly identify kinetically stable proteins in accordancewith the invention is of particular use. A more specific discussion of afew of these many uses is included below for exemplary purposes. Oneskilled in the art can appreciate the many applications of thistechnology are not limited to those described herein.

In one embodiment, the methods of the invention are useful inconjunction with drug discovery. For example, in diseases characterizedby protein misfolding and aberrant protein aggregation (see, Table 1 andassociated references), novel compounds such as small molecules can betested for their ability to impart kinetic stability to a protein thatmay otherwise misfold or aggregate. One example of a drug discoverymethod utilizing the methods of the invention comprises the steps of: a)providing a library of small molecules; b) probing the library with aprotein prone to misfolding and/or aberrant aggregation; c) assaying theproteins complexes to which the small molecules have bound(“protein/small molecule complexes”) for kinetic stability by testingthe ability of the protein/small molecule complexes to resistdenaturation by SDS. Resistance to SDS denaturation may be tested, forexample, by using an SDS-PAGE assay, a fluorescence based assay or a 2DSDS-PAGE assay of the invention. The small molecules that have impartedkinetic stability to a protein to which the small molecule has bound mayserve as the basis for further investigation and drug development.

TABLE 1 Amyloid-Forming Proteins Known to be Associated with DiseaseAmyloido- Precursor Clinical Native genic Protein Syndrome Structurespecies β-protein Alzheimer's Little stable Aβ1–40, disease secondary1–42 structure Serum Amyloid A Secondary Mixed Full length, Systemic(AA) alpha/beta 1–76 Amylodosis Transthyretin Senile Systemic Mainlybeta Full length, Amyloidosis; fragments familial amyloid polyneuropathyIslet Amyloid Type II diabetes Little stable Full length Peptide(amylin) secondary structure Immunoglobulin Primary Systemic Fulllength, Light Chain (light chain) fragments Amyloidosis ApolipoprotienFamilial Amyloid Mainly helix Fragments A–I Polyneuropathy (III) AtrialAtrial Natriuretic Amyloidosis Factor Gelsolin Finnish Mixed 71 residueHereditary alpha/beta fragment Systemic Amyloidosis Cystatin CHereditary Cerebral Amyloid Angiopathy β₂- Hemodialysis- microglobulinrelated amyloidosis Prion Spongiform Mainly helix Full length,Encephallpathies fragments Calcitonin Medullary Little stable Fulllength Carcinoma secondary of the Thyriod structure (Central helix withturns) Fibrinogen Hereditary Renal Mixed Mutant form amyloidosisalpha/beta Insulin Injection- Mainly helix Full length localizedamyloidosis lactadherin Aortic medial unknown Residues amyloid 245–294(Medin) Lysozyme Hereditary non- Mainly helix Mutant form neuropathicsystemic amyloidosis α-Synuclein Parkinson's Little stable Mutant formdisease secondary structure Tau Protein Alzheimer's Little stabledisease secondary structure Superoxide FALS Mainly beta Dismutase bovineCataracts (?) Mainly beta crystallins SH3 domain of the None knownMainly beta Full length p85alpha subunit of bovine phosphatidylino-sitol 3′- kinase (PI3-SH3) Huntingdon Huntington's Polyglutamate ProteinDisease (greater than 39) repeats

In another embodiment, the methods of the invention are useful inconjunction with diagnostic procedures for identifying proteins thathave an increased tendency to misfold and aggregate, resulting indiseases such as those listed in Table 1. The present invention isuseful in quickly identifying proteins in specific cells (by culturingthe cells), or in plasma, that have lost their stability due to mutationor other pathology associated with the disease. One exemplary diagnosticassay comprises the steps of: a) obtaining a protein sample from apatient known to contain a protein whose loss of kinetic stability leadsto disease; b) assaying the protein sample for proteins resistant toSDS; and c) identifying proteins that are not resistant to denaturationby SDS. Those proteins that are not resistant to denaturation by SDS arenot kinetically stable. The loss of kinetic stablility of a proteinknown to be associated with disease may be indicative of the onset orpresence of the associated disease.

In yet another embodiment the methods of the invention are useful inconjunction with prophylactic or therapeutic treatments of disease. Inone example of this embodiment, diseases characterized by the onset ofinstability of certain proteins may be treated by administering to thepatient a kinetically stable form of the same protein which may then becapable interact with defective protein and stabilize or prevent theunstable protein from aberrant aggregation. Therefore, in oneembodiment, diseases characterized by the instability of a proteinassociated with a disease are treated by administering to a patient inneed thereof, a therapeutically effective amount of a kinetically stableform of the protein associated with the disease wherein the kineticallystable form of the protein was identified from a library of proteinsknown to be associated with the disease by assaying the library forproteins that are resistant to SDS denaturation.

In order to investigate the relationship between kinetic stability andSDS resistance, over thirty proteins, including some known to be SDSresistant and/or kinetically stable, were studied (Table 2).

TABLE 2 List of Proteins that Were Analyzed by SDS-PAGE to Assay for SDSResistance 2° No. of Protein Structure Subunits SDS-stable? alcoholdehydrogenase mixed 1 no avidin beta 2 yes beta amylase alpha 1 nocarbonic anhydrase mixed 1 no catalase alpha 1 no chymopapain mixed 1yes chymotrypsin mixed 2 no concanavalin A beta 2 no gamma crystallinbeta 1 no beta glucoronidase mixed 2 no glyceraldehyde-3- alpha 6 nophosphate dehydrogenase hemocyanin mixed 6 no hemoglobin alpha 2 nohyaluronidase alpha 1 no insulin mixed 1 no alpha lactalbumin mixed 1 noluciferase alpha 2 no lysozyme mixed 1 no beta microglobulin beta 1 noneuraminidase beta 1 no papain mixed 1 yes P22 tailspike beta 3 yespectin lyase A beta 2 no rhodanese mixed 1 no ribonuclease A mixed 1 norubredoxin beta 1 no serum amyloid P beta 5 yes streptavidin beta 4 yesCu/Zn superoxide beta 2 yes dismutase transthyretin beta 4 yes triosephosphate alpha 2 no isomerase trypsin beta 1 no urease mixed 2 no

Among these were a few control proteins (streptavidin, transthyretin,and P22 tailspike) that were independently known to be both kineticallystable (via folding experiments) and SDS resistant. SDS resistance wasassayed by comparing the migration on a gel of boiled and unboiledprotein samples containing SDS as shown in FIGS. 2A and 2B. Proteinsthat migrated to the same location on the gel regardless of whether ornot the sample was boiled were classified as not being stable to SDS asshown in FIG. 2B. Those proteins that exhibited a slower migration whenthe sample was not boiled were classified as being at least partiallyresistant to SDS-induced denaturation as shown in FIG. 2A. The slowermigration is a sign of decreased SDS binding, and consequently of alesser overall negative charge of the SDS-protein complex compared tothe fully SDS-bound proteins. Of the proteins tested, eight were foundor confirmed to exhibit resistance to SDS, including Cu/Zn superoxidedismutase, streptavidin, transthyretin, P22 tailspike, chymopapain,papain, avidin, and serum amyloid P (Table 2, FIG. 2A).

To probe the kinetic stability of the SDS-resistant proteins,fluorescence spectroscopy was used (FIG. 3) to demonstrate their slowunfolding rates even in 6.6 M GdnHCl at 20° C. To gather furtherevidence of the kinetic stability exhibited by these proteins undernative conditions, their unfolding rate constants in the absence ofdenaturant were obtained by measuring the unfolding rate at differentGdnHCl concentrations and extrapolating to 0 M (FIG. 4A). The nativestate unfolding rate constants for TTR (17) and SVD (14) were obtainedfrom the literature. The unfolding rate in the absence of denaturantsfor all the SDS-resistant proteins was found to be very slow (Table 2),with protein half-lives ranging from 79 days to 270 years. Thus, thefact that all the SDS-resistant proteins are also kinetically stable,suggest that the latter property may be responsible for the former.

To further test the correlation between kinetic stability and SDSresistance, a group of six proteins was selected that did not exhibitresistance to SDS and analyzed their unfolding behavior in varyingconcentrations of GdnHCl. The group was chosen to represent a variety ofstructural characteristics, and consisted of porcine triosephosphateisomerase (TIM), glyceraldehyde 3-phosphate dehydrogenase (GAPDH),beta2-microglobulin (B2M), bovine alpha-lactalbumin (BLA), concanavalinA (ConA), and yeast alcohol dehydrogenase (ADH). At 6.6 M, the unfoldingof these proteins was too fast to detect with a standard fluorescencespectrophotometer (data not shown). The lack of kinetic stabilityexhibited by these proteins was further demonstrated by their nativeunfolding rates, which ranged from 14 min to 19 h (FIG. 4B, Table 2).

The results support the existence of a correlation between kineticstability and resistance to SDS-induced denaturation. Therefore,SDS-PAGE serves as a simple method for identifying and selectingkinetically stable proteins. This method has the advantage that proteinscan be easily tested for kinetic stability without having to carry outunfolding experiments. Also, only microgram amounts of sample areneeded, and the method is potentially suitable for identifyingkinetically stable proteins present in cell extracts without need forpurification. From an application perspective, this assay has thepotential of being adaptable for various high-throughput applications toenhance the kinetic stability of proteins of interest. This could leadto proteins with greater shelf life and/or decreased tendency toaggregate, consistent with the suggestion that the deterioration of anenergy barrier between native and pathogenic states as a result ofmutation may be a key factor in the misfolding and aggregation of someproteins linked to amyloid diseases (4, 18).

SDS is an anionic detergent, and is a strong denaturant of proteins whenpresent at concentrations above its critical micelle concentration (˜7mM in water) (19, 20). Although there are examples in the literature ofproteins that are not susceptible to denaturation by SDS, it is notclear what chemical-physical property is responsible for thisresistance. It has been shown that there is no correlation betweenthermodynamic stability and SDS resistance (21). Although it has beensuggested that surface charges in a protein can modulate SDS resistance,there is no general effect. For example, very acidic proteins, such aspepsin, will not interact with SDS because of charge repulsion (21).However, proteins with a high ratio of basic to acidic residues on thesurface are not necessarily more susceptible to being denatured by SDS.The ratio of basic to acidic residues was calculated on the eight SDSresistant proteins in the study and a value of 1.2±0.2 was obtained,which is slightly higher than the ratio (0.95) that was calculated forthe average protein based on amino acid composition.

To explore the correlation between SDS-resistance and the structuralrigidity that makes some proteins resistant to proteolytic cleavage, theSDS-resistant and non SDS-resistant proteins were subjected to aproteolytic susceptibility test using the non-specific and aggressiveprotease, proteinase K at the protease: protein ratio of 1:100. As shownin FIG. 5A, the SDS-resistant proteins remained largely intact after 48hours of incubation with proteinase K at 25° C. Only TTR and SAPexhibited a small degree of degradation by proteinase K (FIG. 5). Theremarkable degree of resistance to proteolysis exhibited by thekinetically stable/SDS-resistant proteins is uncommon among proteins andhints at the unusual degree of structural rigidity they posses. Incontrast, of the 25 non SDS-resistant proteins studied, 18 werecompletely degraded and 7 proteins exhibited some degree of proteolyticresistance. A representative group (same as in FIG. 2B) is shown in FIG.5B. These results show that whereas kinetically stable proteins areresistant to proteolytic cleavage, kinetic stability is not arequirement for proteolytic resistance. The different requirements forSDS- and protease-resistance may be due to the non-specific binding ofSDS to unfolded proteins in contrast to the specific binding requirementof proteases. While a protease requires the binding of specificstructural elements to its active site in order to initiate proteincleavage, SDS appears to only require access to the protein's interior.It has been shown that protease-resistance is influenced not only by theexposure of the unfolded chain, but also by sequence determinants withinthe unfolded protein that may caused these proteins to be poorsubstrates for proteolytic degradation (22). Thus, SDS-resistance is amuch more effective probe for identifying proteins with high kineticstability.

The results point to kinetic stability as the molecular basis for theresistance of some proteins to SDS. Since each of the SDS-resistantproteins studied was also found to be resistant to proteolysis byproteinase K, it is proposed that, like in the case of proteolyticsusceptibility, resistance to SDS is linked to the reduced occurrence ofboth local and global unfolding transitions in these proteins. Likeproteases, SDS binding appears to rely on transitions between proteinconformations, moments of weakness in which the protein is susceptibleto SDS binding, and thereby entrapment. Kinetically stable proteins arecharacterized by unusually low structural flexibility (12, 23). Thestructural rigidity of kinetically stable proteins results insuppression of partial unfolding. Furthermore, Truhlar et. al. haveshown that it is not only the barrier towards global unfolding, but alsothe high cooperativity of the unfolding transition of kinetically stableproteins that results in its protease resistance (and presumably SDSresistance) by limiting partial unfolding transitions (24). Thus, thismay explain why unless provided with energy in the form of heat (e.g.through boiling), kinetically stable proteins infrequently assume suchopen conformations under native conditions, and are therefore, resistantto SDS.

A key to understanding kinetic stability in proteins may lie indetermining the physical basis for their structural rigidity, as thisappears to be a common property of kinetically stable proteins (12, 23).Arguably, the most compelling evidence that rigidity may be the keyphysical requirement for protein kinetic stability comes from theobservation that proteins become highly rigid and kinetically stablewhen incubated in very high concentrations of organic solvent (25, 26).In such an environment, the absence of bulk water presumably reduces theenergetic driving force for partial and global unfolding, therebyincreasing rigidity (26). Under normal aqueous environment, kineticallystable proteins may owe their rigidity to the lack of weak points on itssurface where bulk water could penetrate to induce local and globalunfolding. Consistent with this idea, recent work by Machius et. al. hasshown that kinetic stability can be increased by introducing hydrophobicmutations at the surface of protein to stabilize and rigidify regionsthat may be involved in local unfolding (5). Furthermore, strategicallylocated metal ions, disulfide bonds, salt-bridges, and hydrophobicresidues at the surface, may be useful for enhancing the kineticstability of a given protein by serving as molecular “clips” or“staples” to avoid water penetration, resulting in rigid structureswhere partially unfolded states are not sampled under native-likeconditions.

In an attempt to better understand the structural basis for kineticstability, a rudimentary structural analysis was performed on the poolof SDS-resistant proteins based on their structure coordinates obtainedfrom the Protein Data Bank (PDB) (FIG. 6). Each protein was found toexhibit specific stabilizing characteristics, including disulfide bonds(PAP, CPAP, and SOD), oligomeric interfaces (all except PAP and CPAP),and bound metals (SOD). Amino acid composition calculations based on thePDB coordinates likewise yielded no common trend in the amino acidcontent and no consistent deviation from the amino acid compositionfound in natural proteins, implying that no correlation exists betweenkinetic stability and primary structure. However, the presence ofpredominantly β-sheet and oligomeric structure emerged as a commoncharacteristic of most of the kinetically stable proteins studied. It isplausible that the higher content of non-local interactions in β-sheetproteins may allow for higher rigidity than in α-helical proteins.Although clearly not all oligomeric β-sheet proteins are kineticallystable/SDS-resistant (see Table 2), the apparent bias for kineticstability in oligomeric β-sheet proteins may serve to prevent them fromaggregation. When induced to assume an unfolded conformation, β-sheetproteins are particularly susceptible to misfolding and aggregation,potentially leading to protein misfolding diseases (18). It has beenshown that to avoid aggregation natural β-sheet proteins use variousnegative design strategies, such as the placement of loops, β-bulges,prolines, and charged residues at the end of β-sheets (27). Perhapskinetic stability may be another strategy used by nature to minimize thepotential for protein misassembly. It will be interesting to determinehow widespread this property is among other oligomeric and/or β-sheetproteins and whether it correlates with their tendency towardsaggregation. Despite the apparent bias towards β-sheet proteins, theobservation of kinetic stability and SDS-resistance in PAP and CPAP,which are monomeric proteins containing an α- and a β-domain, show thatthese properties are also accessible to other proteins.

In summary, the above results suggest that SDS resistance is a commonproperty of kinetically stable proteins and that SDS-PAGE may be used asa simple assay to probe for kinetic stability in purified proteins orprotein extracts. Without being limited to any scientific theory, theinventors believe that, analogous to proteolytic susceptibility,proteins become vulnerable to denaturation by SDS during their partialand global unfolding transitions. However, due to the non-specificnature of SDS denaturation, it is highly effective in detecting proteinsthat possess high kinetic stability. Together, these results providecompelling support to the idea that kinetic stability is a commonproperty of rigid protein structures. The identification of a simple andeffective assay for kinetic stability assay in accordance with theinvention provides the opportunity to accumulate a larger database ofkinetically stable proteins, thus paving the way for further studiesgeared towards understanding the relationship between kinetic stabilityand protein structure.

EXAMPLES Example 1

SDS-polyacrylamide Gel Eectrophoresis (SDS-PAGE) Assay. Lyophilizedproteins were obtained from Sigma (papain (PAP), chymopapain (CPAP),avidin (AVD), and superoxide dismutase (SOD)) and Calbiochem(streptavidin (SVD), serum amyloid P (SAP), and transthyretin (TTR)).Salmonella phage P22 tailspike (TSP) protein was a gift from J. King(MIT). All the remaining proteins (Table 2) were obtained from Sigmawith the exception of catalase, which was purchased from Calbiochem.Stock solutions (1 mg/mL) of all proteins studied except papain,chymopapain, and P22 tailspike protein were made using 10 mM sodiumphosphate buffer (pH 7.0) (PB). Stock solutions (1 mg/mL) of papain andchymopapain were prepared with 25 mM2-amino-2-hydroxymethyl-1,3-propanediol (Tris), 1 mM EDTA (pH 5.3). Thestock solution of the P22 tailspike protein was 0.8 mg/mL in 50 mM Tris,2 mM EDTA (pH 7.6). All electrophoresis samples contained ˜5 μg proteinand 1% sodium dodecyl sulfate (SDS) in 0.125 M Tris (pH 6.8). Proteinsamples were unheated or boiled for 10 min. prior to analysis bySDS-polyacrylamide gel electrophoresis (SDS-PAGE), using 15% AcrylamidePager Gold precast gels (Cambrex), and 0.1% SDS in Tris/Glycine buffer(pH 8.3) as running buffer. The gels were then stained using CoomassieBlue.

Example 2

Proteolysis. For limited proteolysis experiments the concentration ofthe proteins was determined by weighing the lyophilized protein on amicrobalance. Each proteolysis reaction mixture contained about 0.5mg/mL protein and 5 μg/mL proteinase K (Fisher Scientific) in 25 mMTris, 1 μM EDTA (pH 8.3), and was incubated at 25° C. for 48 hours. Thereaction was stopped with a solution of 2.5 μM phenylmethylsulfonylfluoride, 4% SDS in 0.125 M Tris, 3.4 μM 1,4-Dithio-DL-threitol (pH6.8)). Samples were boiled and gel electrophoresis was performed using16% acrylamide Novex precast gels (Invitrogen). Running buffer was 0.1%SDS in Tris/Tricine buffer (pH 8.1).

Example 3

Fluorescence. Unfolding kinetics induced by guanidine hydrochloride(GdnHCl) were monitored using an F-4500 fluorescence spectrophotometer(Hitachi, Danbury, Conn.). The concentration of GdnHCl was determinedusing an Abbe Mark II refractometer (Leica, Buffalo, N.Y.). Proteinsolutions (0.05 mg/mL) in 25 mM PB, 0.20 M sodium chloride (pH 7.2) weretreated with GdnHCl solution made using the same buffer to a finalconcentration of 6.6 M. The excitation/emission wavelengths used were:275/350 nm (B2M), 275/360 nm (BLA, ConA, GAPDH, TIM), 280/320 nm (ADH),295/360 nm (CPAP, TTR), 295/350 nm (PAP), 295/340 nm (SAP, TSP), 280/330nm (SOD), 295/333 nm (SVD), and 280/340 nm (AVD). Kinetic traces wereanalyzed by fitting to a sum of exponentials.

Example 4

2D-SDS PAGE

In recent years, various methods have been developed for high throughputproteomic analysis. The first step of such techniques usually consistsof two-dimensional gel electrophoresis (2DE), a powerful method for therapid separation of thousands of proteins in one experimental step. In2DE, the first dimension usually separates proteins based on theirisoelectric point, followed by an SDS-PAGE separation according to MW.Although the traditional 2DE method provides a powerful means for theseparation of thousands of proteins, it is time-consuming, requiresexpensive gel analysis software for interpretation, and can beassociated with decreased reproducibility. In addition, variousexperimental issues, including the requirement for urea in the firstdimension run and the sensitivity of the first dimension strip to heat,make the traditional 2DE methodology unsuitable for selectingkinetically stable proteins from complex mixtures. In order to addressthese issues, the inventors have developed a diagonal 2DE method inwhich an SDS-PAGE separation is performed in both dimensions of theanalysis. Protein mixtures are first separated according to molecularweight on a narrow strip of gel. The gel strip is then incubated inSDS-PAGE treatment buffer while briefly immersed in a boiling water bathbefore being transferred onto a larger gel for the second dimension run.By using this methodology, the inventors are able to translate ourSDS-resistance assay to 2D SDS-PAGE for the rapid and high throughputdetermination of kinetic stability.

Most proteins are denatured by SDS even without heating, and thusmigrate the same distance in both dimensions of our analysis, resultingin a diagonal line of spots across the gel (FIG. 7A). However,SDS-resistant proteins will travel a shorter distance in the 1stdimension. In the 2nd dimension, these SDS-resistant proteins willbecome denatured because of the heating step and will have the expectedSDS-PAGE migration, consequently falling off the diagonal. FIG. 7B showsthe behavior of the SDS-resistant proteins transthyretin, streptavidin,and superoxide dismutase. Due to this difference in migration ofnon-SDS-resistant and SDS-resistant proteins on gel, kinetically stableproteins can easily be selected from a mixture of proteins. This can beseen in FIG. 7C for a mixture of 3 SDS-resistant proteins and 9 standardnon-SDS resistant proteins. FIG. 8 illustrates how this method can beused to identify kinetically stable proteins from a cell lysate of E.coli. The migration of SDS-resistant proteins below a diagonal canprovide a simplified means of identifying which proteins are kineticallystable without the need for expensive computer analysis software. Inaddition, the analysis time is considerably reduced with our method incomparison with traditional 2DE (3 hours versus 2–3 days). Furthermore,the simple protocol associated with this method will make it much easierfor students and professionals to utilize this fast means of identifyingkinetically stable proteins from a mixture of proteins.

The novel diagonal 2DE assay of the invention provides a means ofhigh-throughput identification of kinetically stable proteins fromcomplex mixtures. The method has many potential applications in thefields of basic bioscience, pharmaceuticals, medical diagnostics, andengineering. Determination of the kinetically stable proteomes ofvarious organisms can yield important information about the biophysicalbasis for kinetic stability and evolution of this characteristic inproteins from different species. There is also potential for adaptationof the method for selecting kinetically stable mutants of a givenprotein from mixtures of mutants for applications in pharmaceuticals andindustrial catalysis. A particularly important application of the methodinvolves evaluating the kinetically stable proteome of healthy humanplasma and comparing it to that associated with various diseases inhopes of finding important biomarkers for the early diagnosis of thesediseases in humans. Combination of this approach with a Western-blottingprotocol for specific protein quantification could make it a valuablediagnostic tool for disease detection.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

REFERENCES

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1. A method for identifying SDS resistant proteins in a samplecomprising the steps of: a) electrophoretically separating thecomponents of a sample according to weight on a strip ofSDS-polyacrylamide gel in a first dimension; b) incubating the gel fromstep (a) in SDS-PAGE treatment buffer which is healed to boiling with awater bath; c) transferring the gel from step (b) to a second, larger,SDS-polyacrylaniide gel for electrophoretic separation in a seconddimension; and d) identifying the SDS resistant proteins on the seconddimension gel wherein said kinetically stable proteins are thoseproteins which run off the diagonal of the gel.
 2. The method of claim 1wherein the sample is plasma from a patient.
 3. The method of claim 1wherein the sample is a protein library.
 4. The method of claim 3wherein the protein library is produced or generated by one of themethods selected from the group consisting of rational protein design,protein genesis, directed evolution, site-directed mutagensis and geneshuffling.
 5. The method of claim 3 where the protein library compriseschirneric or mutated proteins.